Gattuso:RNA extraction

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To 500 ml cultures of decalcifying cells add 11 ml of 0.1 M HCl to bring teh final concentration to 2.2 mM.
Swirl the flask and note the clearing of teh culture. After adding the HCl, check if teh pH has dropped from 8.0 to 5.0 using pH strips.
After 1 min, quikly neutralize the solution by adding 14.3 ml of 0.1 M NaOH (final concentration of 2.8 mM). This serves to restore the culture to a neutral pH of 8.0.

Haversting cells
Centrifuge at 10000g for 10 min at RT.
Do not place the cells on ice as this will cold shock the cells and activate specifics RNAses within the cell

1- if the RNA is to be extracted later:
Resuspend the cells in 100% ethanol and pellet the cells by centrifugation before storing at -20°C.

2- if teh RNA is to be extracted immediatly
Resuspend and combine the cells in a disposable centrifuge tube using centrifugation.

RNA extraction

1- Deep the tube in liquid nitrogen few minutes

2- Grind the cells into fine powder using a grinder adapted to the size of the tube.

3- Add 500 µl of extraction buffer and vortex for 30s. vent to release any pressure built up.